In many apoptotic paradigms, a stress signal is delivered to the mitochondria (mitos) to induce release of apoptotic factors from the intermembrane space to the cytosol. These factors trigger execution of a program of cell death. In the cell, mitos often show coordinated activation of the individual organelles, giving rise to Ca 2+release waves and depolarization waves that depend on intermitochondrial communication. However, it remains elusive whether mitos interact with each other to establish coordinated release of apoptotic factors. Our principal hypothesis is that local intermitochondrial signaling is utilized to synchronize the response of individual organelles during apoptosis. We propose that several factors released by the mitos, such as pro-caspases, Ca 2+ and reactive oxygen species (ROS) may serve as a lateral signal that promotes recruitment of neighboring mitos to the apoptotic machinery. Furthermore, we propose that mitos release a novel factor, termed as mitochondrial permeabilization inducing factor (mPIF) that expands permeabilization to other mitos. Finally, we propose that intermitochondrial signaling may be particularly important in apoptosis of cells that are rich in mitos (e.g. muscle and liver). In the previous project period, we have demonstrated that calcium signal propagation to the mitos may trigger apoptosis. We have also shown that calcium release from mitos that undergo membrane permeabilization is important for the membrane permeabilization of the adjacent mitos, providing an example of the regenerative mechanisms that may underlie propagation of the mitochondrial death waves. Our preliminary studies have also shown coordinated recruitment of the mitos during truncated Bid (tBid)-induced mitochondrial permeabilization. Significantly, the tBid-lnduced wave of mitochondrial membrane permeabilization is not dependent on Ca. Furthermore, from mitos we have extracted a heat-sensitive and soluble factor that induces mitochondrial membrane permeabilization. To test our hypotheses, we will develop several high resolution fluorescence imaging methods to study mitochondrial function in single cells and will use single channel electrophysiology and a range of fluorometric, biochemical and molecular techniques. Dissection of the mechanisms of local intermitochondrial signaling is critical for understanding the operation of the apoptotic machinery and in turn, it is a key component in elucidating the regulation of a wide range of physiological and pathological processes that depend on apoptotic cell death.